In biomolecular research and clinical diagnostics it is both important and a challenge to separate and identify rare cells, bacteria, virus, and biomarkers (e.g. DNA, RNA, antibodies, other proteins, etc.) in complex fluid samples like blood, plasma, serum, saliva, and urine. Additionally, the advent of bio-nanotechnology has led to numerous drug delivery approaches that involve encapsulation of drugs and imaging agents within nanovesicles and nanoparticles. Such approaches mean it will now also be important to identify and separate residual nanovesicles and nanoparticles that remain in the blood stream. A variety of physical, electronic, and biological techniques and mechanisms can be used for sample preparation and isolation of specific cells, nanovesicles, and biomolecules from complex samples like blood. These techniques and mechanisms include centrifugation, gel filtration, affinity binding, DC electrophoresis, and various combinations incorporated into lab-on-a-chip, microfluidic devices, and sample-to-answer systems.
Many of these conventional techniques (or combinations) are relatively time consuming processes that are not without problems and limitations. In particular, the isolation of rare cells (cancer cells, fetal cells, and stem cells), low numbers of bacteria and virus or very low numbers of specific antibodies, proteins, enzymes, DNA, and RNA molecules, still remains difficult. In the case of clinical diagnostics, rare cell and biomarker detection may also be limited by sample size; i.e., only a relatively small amount of blood may be drawn from very ill patients, the elderly and infants. Thus, sample preparation processes that are inefficient or require high dilution of the original sample often fail or are unreliable for isolating cells and other disease-related markers at lower concentration ranges. This is in particular a problem for early detection of cancer, residual disease, fetal cells/DNA/RNA in maternal blood, bacteria and virus in blood (septic infection), and the detection of low numbers of pathogens (e.g. bacteria, virus, etc.) and bioterror agents in large volumes of air, water, or in food stuffs.
Alternating current electrokinetic techniques that involve the use of AC fields to manipulate cells and nanoparticles offers some particularly attractive mechanisms for the separation of cells [see References 2-5], biomarkers (DNA [Ref. 5-8], proteins [Ref. 9], etc.), and ultimately drug delivery nanovesicles [Ref. 10]. These techniques can be broken down into three distinct phenomena: (1) AC electroosmosis, which is surface fluid flow due to the surface charge on an electrode; (2) electrothermal flow, which is bulk flow in solution due to thermal gradients produced by the electric fields; and (3) dielectrophoresis (DEP), which is an induced motion of particles produced by the dielectric differences between the particles and media in an AC electric field [Ref. 10]. Unfortunately, most conventional forms of DEP and related electrokinetic effects have problems that limit the usefulness of these technologies for clinically relevant sample preparation and diagnostics.
First, efficient DEP separations in terms of speed and control of selectivity usually have to be carried out at relatively low conductance on the order of <10-100 mS/m [Ref. 11]. Additionally, the ability to isolate the desired entities/analytes such as nanoparticles or DNA biomarkers in the positive or DEP high field regions (usually around or on the electrodes) becomes more difficult as the solution ionic strength increases and the conductance becomes greater than 10 mS/m. Thus, biological samples such as blood or plasma that have ionic strengths in the 100-200 mM range (conductance ˜500-1000 mS/m) must be significantly diluted and/or processed before DEP separations can be carried out [Ref. 13, 14]. This alone often limits the usefulness of DEP for clinical diagnostics involving the detection of rare cells or low numbers of biomarkers. In cases where a sample (one ml blood) has to be diluted 100 to 1000-fold, now means that a very large sample volume must be processed, which can be prohibitively time consuming. If cells are first concentrated by physical mechanisms such as centrifugation or filtration and then are diluted into low conductance buffers, these processes are not only time consuming, but also costly and cause considerable perturbation to the sample. In the case where DEP might be used for stem cell separations, dilution into low ionic strength, less physiological-type buffers may result in perturbation of sensitive stem cells and may affect their further differentiation. The isolation of DNA, RNA, and protein biomarkers from blood is also important for future clinical diagnostics, in particular for monitoring cancer chemotherapy [Ref. 15], residual disease [Ref. 16], and early detection of cancer [Ref. 17].
While DEP has been used for the isolation of DNA and proteins, problems and limitations do exist in using DEP to carry out the detection of DNA in blood. The first problem again is the need to dilute and/or process the blood sample before DEP analysis. In the case of clinically relevant cell-free circulating DNA and RNA biomarkers in blood, finding and measuring the amount of DNA/RNA, its size and base composition (mutations and polymorphisms) is important [Ref. 17-19]. Sample processing that involves or requires centrifugation, filtration, and washing procedures can cause the release of DNA molecules by normal cells that are damaged or lysed in the process, as well as shear the clinically relevant DNA into smaller fragments. The release of extraneous DNA fragments and processing damage to the clinically relevant DNA greatly compromises and limits the diagnostic value of using such procedures. Such sample processing is also highly inefficient, and up to 60% of the DNA and over 90% of the RNA in the blood can be lost during the procedure [17].
A second problem area is that most DEP separation devices that have been used for DNA, protein, and nanoparticle separations use either polynomial gold microelectrodes created with a very small separation (6 μm or less) between them to serve as particle traps; or use castellated gold microelectrode arrays with 6-8 microns or less separation between them [Ref. 18-19]. These gold microelectrode array devices are usually fabricated by sputtering gold unto a glass substrate material. There are also a number of DEP approaches involving the use of nanoelectrodes [20]. The problem with these approaches are that the arrays have intrinsically low throughput, since the actual space to capture DNA or other biomolecules is relatively small and the electric field effect is significantly reduced when distance from the nanoelectrode increases (e.g. >10 nm). If this type of device is scaled for sample preparation (e.g., to process 1-10 ml of blood), the actual sample area that can be interrogated by the limited DEP field near the electrodes means that most of the DNA will be missed, or an extremely long sample processing time would be required. If the device is designed to constrict the liquid flow so as to pass within ten's of nanometers of the nanoelectrodes, then the processing time is again extremely long or a massively large (x-y dimension) device would be required. A variety of other problems exist including uncontrolled fluidic eddy currents due to other electrokinetic effects and osmotic forces. In other DEP applications, arrays that utilize circular platinum microelectrodes (50 μm to-80 μm diameter) with about 200 um spacing and over-coated with a porous hydrogel have also been used to carry out the DEP separation of bacteria from blood, and for the separation of cancer cells [Ref. 13, 14]. Again, for these DEP separations, the blood sample was centrifuged and a small fraction of the cells were re-suspended in a low ionic strength buffer [Ref. 13, 14, 24-26].
A third general problem for AC electrokinetic techniques is often that the resulting sensitivity versus specificity ratios are not sufficiently high for carrying out important or clinically relevant separations. For cell separations using dielectrophoresis (DEP), carrying out efficient rare cell separations with ratios of one in a million is difficult. Because many early disease diagnostics require rare cell or low level biomarker detection, it is important to be able to improve sensitivity versus specificity ratios as much as possible. In general, most DEP devices are not scaled properly to deal with the clinical reality of rare cell or low level biomarker isolation and detection, where a relatively large sample of from 1-10 ml of blood might be necessary for simple statistical reasons. When DEP device are designed for large samples, they are usually inefficient and unable to operate at high conductance conditions, and thus require further sample dilution.
A fourth problem for AC electrokinetic techniques is carrying out efficient (low loss) and highly selective separation processes in complex biological samples (e.g. blood, plasma, serum, etc.) for analytes and biomarkers which include; rare cells, bacteria, virus, DNA, RNA and proteins where all the entities might have 2-3 orders of magnitude difference in size range, and it is still necessary to achieve an efficient separation between entities that are more similar in size and composition. One important example is the separation of DNA nanoparticulates (20-50 kb), high molecular weight DNA (5-20 kb), intermediate molecular weight DNA (1-5 kb), and lower molecular weight DNA (0.1-1 kb).
The final and most serious problem for AC electrokinetic (DEP) devices and techniques is the introduction of electrochemistry that becomes more pronounced in higher conductance solutions (>100 mS/m), at lower AC frequencies (<20 kHz) and at higher voltages (>20 volts pt-pt). As will be shown in the Detailed Description section of this document, such electrochemistry can cause a number of adverse effects including bubbling, heating, fluidic turbulence, electrode degradation, and destruction of labile analytes. These adverse effects greatly limit the overall DEP device performance, prevent the accumulation, isolation, and detection of specific entities (cells, nanoparticles, DNA and proteins) from occurring in the DEP high field regions, and interfere with the isolation of cells and analytes into the DEP low field regions.
Other types of AC electrokinetic devices have been used to separate cells and nanoparticles, but have not proved viable in high conductance solutions. One of the most convincing arguments for the non-viability of AC electrokinetic and DEP devices is the fact that unlike DC electrophoresis, which has widespread use in biological research and clinical diagnostics, DEP has not been used for any practical applications. It would be desirable to perform dielectrophoresis with high performance characteristics that allow separations in high conductance biological samples and buffers.